Tropomyosin (TM) is considered to be a major allergen of crustaceans and shellfish. To address the increasing risk of crustacean and shellfish allergy, in this study, TM enrichment from shrimp (Litopenaeus vannamei) was used as the immunogen of BALB/c mice. A monoclonal antibody (Mab) cell line 2F8 against TM allergen of Litopenaeus vannamei was obtained by hybridoma cell fusion techonology. The immunological properties of Mab 2F8 including titer, specificity, antibody isotype and binding region were characterized using ELISA, Western Blot and bioinformatics. Moreover, its application feasibility was discussed using a sandwich colloidal gold test strips constructed by Mab 2F8 and rabbit polyclonal antibody specific for TM. ELISA and Western Blot results showed that the antibody subtype of Mab 2F8 was IgG1, with an antibody titer of 1.28×10⁵ and an IC₅₀ value of 20.21, indicating a high affinity to TM. It was found that the purified Mab 2F8 had specific reaction with the TMs in crustaceans, shellfish and squid. Three potential binding regions in amino acid sequence AA_0-31, AA_38-43 and AA_58-67 of TM were found according to bioinformatics analysis techniques. Detection results by test strips showed that 5 ng/mL TM (equivalent to 12.5 μg/kg) of white prawn shrimp could be visually detected within 10 min, showing that the obtained Mab 2F8 could be used for the detection of TM in the muscle of crustaceans and shellfish. Our study indicated that the purified 2F8 Mab had high affinity and good application value, which could be used for identification and detection of allergen TM in shrimps, crabs, shellfish and squid. This study provides important biological materials for the identification or detection of allergen TM and technical support for the development of rapid detection kit of TM in the future.